Hemophagocytic lymphohistiocytosis following pembrolizumab and bevacizumab combination therapy for cervical cancer: a case report and systematic review

Background Programmed cell death protein 1 (PD-1) checkpoint inhibitors such as pembrolizumab are novel therapeutics used to treat various advanced malignancies. Immune-related adverse events are common, among the most serious of these toxicities is hemophagocytic lymphohistiocytosis (HLH), which is a life-threatening disorder of unbridled immune activation but has not been properly established. Methods We have procured the first case of hemophagocytic lymphohistiocytosis as an aftermath of treatment with pembrolizumab from the Sir Run Run Shaw Hospital, Zhejiang University, China. In a pursuit to enhance the understanding of this condition, a comprehensive systematic review was performed encompassing all reported instances of ICI-associated Hemophagocytic lymphohistiocytosis within the realms of PubMed and Embase databases. Results We detail the recovery of a cervical cancer patient with a history of psoriasis who developed HLH after combined pembrolizumab and bevacizumab treatment. Remarkably, tumor lesions exhibited substantial and sustained regression. From an analysis of 52 identified Immune Checkpoint Inhibitor (ICI)-related HLH cases, we discovered that HLH often occurred within the first two treatment cycles and approximately 20% of these patients had a history of autoimmune-related diseases. Despite a 15% mortality rate, the majority of patients experienced positive outcomes. Notably, in instances of recovery from HLH, 80% showed positive tumor outcomes. Even after discontinuation of ICI treatment, tumor control persisted in some cases. Conclusion We identified the first case of HLH caused by ICI treatment in cervical cancer and summarized the possible occurrence factors of these cases, the treatment outcomes of HLH, and the impact on tumor outcomes. Supplementary Information The online version contains supplementary material available at 10.1186/s12877-023-04625-3.


Background
Cancer immunotherapy using immune-checkpoint inhibitors (ICIs) has emerged as a cornerstone treatment for various cancers.However, by eliminating the physiological inhibitory control that moderates T-cell activation, ICIs may cause T-cell hyperactivation and immune-related adverse events (irAEs) [1], one rare but serious irAE is hemophagocytic lymphohistiocytosis (HLH).HLH is a severe condition caused by immune activation and dysregulation, potentially leading to excessive pro-inflammatory cytokine secretion, rapid tissue destruction, multi-organ failure, and death [2].Although the incidence of ICI-associated HLH is very low, with the World Health Organization's VigiBase pharmacovigilance database reporting only 5.7% of all HLH cases as potentially ICI-associated [3], the Society for Immunotherapy of Cancer (SITC) includes HLH in lethal irAE but does not provide specific treatment recommendations [4].
In the phase I/II CheckMate 358 trial (NCT02488759) [5] and KEYNOTE-158 (NCT02628067) [6], large clinical trials confirming the safety of ICIs in cervical cancer, ICIs have been widely used for cervical cancer treatment.However, no serious hematological adverse reactions due to ICIs in cervical cancer have been identified recently, and we report the first case of HLH attributed to ICIs in cervical cancer.This finding has not been previously documented in the literature or pharmacovigilance databases.
To better understand the biomarkers of ICI-associated HLH, clinical features, treatment strategies, characteristics of deceased cases, and the potential impact of HLH on tumor outcomes, we gathered all cases that met the HLH-2004 [7] or HScore > 169 criteria [8].(Supplemental data 1) for ICI-related HLH.In total, we analyzed 52 confirmed ICI-related HLH cases by comparing our collection with data from two WHO pharmacovigilance databases.Our review revealed an increasing occurrence of ICI-related HLH in various cancer types and ICI classifications, a high number of cases with a history of autoimmune disease in HLH occurrence, autoimmune encephalitis as a significant cause of death, and the potential for an exceptional oncologic response to treatment if recovery from HLH is achieved.

Case report
The clinical case of HLH was encountered by the authors in their clinical practice at Sir Run Run Shaw Hospital, Zhejiang University, China.Clinical data were gathered through review of the electronic patient journal.Data were visualized using GraphPad Prism V.9.4.0.

Systematic review
We conducted a systematic search using medical subject headings (MESH) in PubMed and Embase databases up to April 2023 to explore the keywords "Hemophagocytic Lymphohistiocytosis" and "immune checkpoint inhibitors".Search strategy free words and grid terms are as follows: ("Immune checkpoint inhibitor"OR "anti-PD1"OR"anti-PD-L1"OR "anti-CTLA4""avelumab" OR "atezolizumab" OR "cemiplimab" OR "durvalumab", OR "ipilimumab" OR "nivolumab" OR "pembrolizumab"OR "camrelizumab") AND ("Hemophagocytic Lymphohistiocytosis" OR "HLH" OR "Macrophage Activation Syndrome"OR "lymphohistiocytic syndrome"OR"hemophagocytic syndrome").Through manual retrieval of the references included in this study, other relevant studies that were not found in the retrieval process in databases were found shows the literature acquisition flow chart for the meta-analysis.(Supplemental data 2) Inclusion criteria for abstract review were assessed independently by two reviewers (XHJ and CYZ) and defined as follows:(1) report on HLH or related systemic syndromes on human patients with malignant tumors.(2) report use of ICI as the primary therapeutic agent before the onset of HLH.(3) case series or case reports, that is, no randomized controlled trial.( 4

Case report
We report the case of a 73-year-old Chinese woman with a medical history of psoriasis.Pathological examination suggested moderately differentiated squamous cell carcinoma, and a comprehensive PET-CT scan revealed a 72*52 mm soft tissue mass in the cervix with lesions invading the uterus upwards and the upper third of the vagina, increased FDG uptake with a maximum SUV of 21.33, several mass lymph nodes in the mediastinum, right pulmonary hilar, and pelvis, increased FDG uptake with a maximum SUV of 15.44.Leading to a diagnosis of stage IVB cervical squamous carcinoma.NGS testing indicated PD-L1: TPS = 71%-80%, Microsatellite stable, TMB = 8.96Muts/Mb, and mutations in AKT1, KMT2C and NF1.The patient, initially undergoing one course of paclitaxel and cisplatin treatment, transitioned to pembrolizumab and bevacizumab therapy due to fatigue.
Seven days after the first cycle, the patient was readmitted to the hospital with fever, dry mouth, and right iliac pain, with a maximum temperature of 38.5 degrees Celsius.Physical examination showed no congestion or swelling in the throat, slight breathing sounds in both lungs, no obvious dry or wet rales and no obvious tenderness or rebound pain in the entire abdomen.Following abdominal CT scans, no sign of active inflammatory lesion was observed.Results from laboratory blood tests revealed that leukocyte counts were within the conventional normality limits.Both the absolute quantitative count and its corresponding proportion of neutrophils were within the normal range.The hypersensitive C-reactive protein level was raised to 23.9 mg/L.Comprehensive physical examination, CT scans, and laboratory examination still cannot completely rule out fever caused by bacterial infection.The fever subsided after acetaminophen and anti-infective symptomatic treatment with levofloxacin, but dry mouth and fatigue symptoms persisted.Two days later, the patient developed fever again with a maximum temperature of 38.8 degrees Celsius, and routine blood tests indicated leukocytes at 0.8 × 10 9 /l, neutrophils at 0.03 × 10 9 /l, hemoglobin at 103 g/l, and platelets within the normal range.The patient was given imipenem for preventing infection and granulocyte colony-stimulating factor for leukocyte therapy.On the fifth day of hospitalization, the patient developed a rash (Fig. 1), and a reexamination showed neutrophil counts close to 0/mm3, hemoglobin levels at 89 g/l and IL-6 at 28.4 pg/ml.Tests for COVID-19, EBV DNA, respiratory viruses, CMV, rubella, and herpes viruses all returned negative results.
We strongly suspected immune therapy-related granulomatous deficiency with fever and administered intravenous immunoglobulin and methylprednisolone shock therapy, followed by a bone marrow biopsy.The biopsy revealed a number of hemophagocytic cells (Fig. 2), sCD25 (pg/ml) at 7263 (< 6400), NK cell activity (%) at 11.27 (> 15.11), and an ultrasound that did not show splenomegaly, meeting the 6 of the 8 diagnostic criteria in HLH-2004.Upon evaluating both pembrolizumab and bevacizumab using the Naranjo Score (refer to Supplementary data 3), pembrolizumab registered a score of 6, while bevacizumab received a 3. Considering the rarity of HLH as a complication of ICI therapy and the lack of evidence linking anti-vascular therapy to HLH onsetthough it may intensify ICI effects-we deduce that pembrolizumab primarily induces HLH, with bevacizumab potentially worsening the syndrome.The patient was diagnosed with hemophagocytic lymphohistiocytosis and treated with methylprednisolone, posaconazole, and caspofungin due to test results indicating a G-test value of 30.70 pg/mL and a GM-test value of Aspergillus galactomannan at 3.230ug/L.After a week of treatment, the patient's body temperature returned to normal.
In the second week, the patient experienced fever again, and a pathogen NGS test suggested Enterococcus faecalis infection, leading to the addition of tigecycline to her treatment.Three days later, the fever disappeared and routine blood tests showed that leukocytes and hemoglobin levels had mostly returned to normal ranges (Fig. 3).The patient was discharged with oral medications methylprednisolone tablets, linezolid tablets, and posaconazole, and fever, rash, hip pain, and bone marrow suppression did not recur.Within six months after the patient's last ICI treatment, the patient did not undergo any antitumor therapy, and the latest follow-up results still showed significant and durable tumor regression without recurrence.

Systematic review
We analyzed a total of 40 articles, excluding 2 articles from the pharmacovigilance database [3,9] in the WHO repository for separate discussion due to insufficient Fig. 1 Physical findings.Macular rash observed on the bilateralis buttock, thighs and cervico-thoracic region clinical information for diagnostic evaluation.From the remaining 38 literature reports, we identified 52 cases of HLH associated with ICIs.The primary characteristics of these cases are summarized in Table 1.Our findings reveal that 28 patients were male and 24 were female, with a median age of onset at 60 years (ranging from 2 to 81 years), indicating that HLH can occur at any age.As noted in the WHO pharmacovigilance database, the age of onset varied considerably, from a young child of 2 years to an elderly individual of 101 years.Immunotherapy was administered for lung cancer (n = 13), melanoma (n = 20), bladder cancer (n = 2), kidney cancer (n = 3), and leukemia (n = 2) (Fig. 4B), with proportions comparable to those in the pharmacovigilance database [3].However, no similar cases were reported for cervical cancer.
Most cases involved single ICI therapy.However, among the combined anti-tumor therapies, two were combined with radiotherapy, two leukemia cases with allogeneic stem cell transplantation, two cases with dabrafenib + trametinib, two cases with anti-angiogenic therapy (cabozantinib and bevacizumab, respectively), and three cases with carboplatin, paclitaxel, and pemetrexed chemotherapy alongside immunotherapy.The number of ICI treatment cycles before the diagnosis of HLH ranged from 1 to 14, but incidences tended to decrease as the number of cycles increased.Excluding cases with missing information, HLH presented in 62% of all cases during the first 1-2 cycles of treatment.The time from the last Of the case reports with previous history, 8 (20.5%) patients had previous autoimmune related diseases, such as rheumatoid arthritis, immune thrombocytopenic purpura, Sjögren's syndrome, immune thyroiditis, psoriasis, sarcoidosis, or positive antinuclear antibodies, which is significantly higher than the prevalence of autoimmune diseases in the general population.
Upon follow-up, we observed an interesting phenomenon consistent with our case: 8 cases reported that ICI-induced HLH produced significant and long-lasting responses to the tumor.After the last ICI treatment, the tumor continued to shrink or remained stable on multiple reviews, even without any other anti-tumor therapies.

Discussion
Hemophagocytic lymphohistiocytosis (HLH) is a severe, hyperinflammatory syndrome triggered by aberrantly activated macrophages and cytotoxic T lymphocytes (CTLs) [51].In adults, it frequently arises in contexts like untreated hematologic malignancies, chronic rheumatic diseases, or conditions of immunosuppression.The pathogenesis of HLH involves excessive activation of CTLs and their consequential depletion of IL-2, potentially leading to regulatory T cell (Treg) dysfunction [52,53].This cascade is pivotal in HLH development.Integral to ICI-based cancer immunotherapy is the simultaneous inhibition of Tregs and activation of CTLs [54].ICIs enhance T-cell activation and proliferation while impairing Treg cell functionality.By inhibiting immune checkpoint molecules, ICIs prevent tumor cells from evading immune detection [54].This same mechanism, however, disrupts peripheral T-cell tolerance, fostering the rapid diversification and clonal expansion of potentially toxic cells, which can culminate in hyperinflammation and autoimmunity [55,56].Furthermore, in HLH, CTL activation is perpetuated by a macrophage/monocyte expansion loop [51].Studies in mouse and human cancers indicate that tumor-associated macrophage PD-1 expression inversely correlates with its phagocytic efficacy against tumor cells.Accordingly, PD-1/PD-L1 blockade has been observed to enhance macrophage-mediated phagocytosis of tumor cells [57].These studies offer insights into the specific mechanisms of HLH associated with immunotherapy and confirm that immunotherapyrelated HLH is a genuine and serious complication.
ICI becomes increasingly popular in treating various cancers, the number of immune-related adverse event (irAE) reports has also grown exponentially [58].For instance, there were only 38 cases of ICIassociated HLH in VigiBase before 2019, but the latest study showed 177 cases in VigiBase.Our brief review suggests that ICI-associated HLH can occur across a broader range of patient populations and cancer types, including the first-ever reported case in cervical cancer, which we found outside the database.The latest Vig-iBase data on age at onset aligns with the findings from our literature collection, indicating a median age of onset around 60 years.Furthermore, very young onset cases were also found in both our systematic review and the database, with patients as young as two years old.In our analysis, melanoma and lung cancer have emerged as the most common cancer types associated with ICIrelated HLH.The predominance of these cancers in HLH cases can be linked to the early approval of ICIs for their treatment, leading to a more extensive pool of data and reported cases.Therapies like pembrolizumab, nivolumab, and their combination with ipilimumab, predominantly used in melanoma and lung cancer, have been well-documented in this regard.However, as the spectrum of approved ICIs expands, an increase in HLH incidence is being observed in other cancers.This trend reflects the evolving landscape of ICI usage and its associated irAEs.Notable among these are various types of acute myeloid leukemia (AML) [45], as well as previously undetected cases in squamous cell skin cancer [44], Kaposi sarcoma [35] and other cancer types.While melanoma and lung cancer currently F female, M male, HLH hemophagocytic lymphohistiocytosis, ICI immune checkpoint inhibitor have a higher number of HLH cases due to their early inclusion in ICI therapy, the rising number of HLH cases in cancers like AML, squamous cell skin cancer, and Kaposi sarcoma, as seen with newer ICIs like camrelizumab and avelumab, indicates a broader oncological concern.Identifying specific cancer types or ICI agents that are more prone to inducing HLH remains a challenge, necessitating further research to clarify these associations and improve our understanding and management of ICI-related HLH across different cancer scenarios.
Our case involved the combination of bevacizumab for anti-angiogenic therapy, and prior to this, there were two reported cases of HLH resulting from ICI combined with anti-angiogenic therapy.One of these previous cases suggested that the development of HLH was closely linked to anti-angiogenic therapy [26].Several studies using the FDA adverse event reporting system (FAERS) database        have demonstrated that bevacizumab combined with PD-1 monoclonal antibody increases the risk of serious adverse effects such as fever, physical condition deterioration, thrombocytopenia, bone marrow failure, and neutropenia in oncology patients [59,60].Regarding specific mechanisms, VEGF can inhibit T cell function, increase Tregs and MDSCs, and hinder the differentiation and activation of DCs [61].However, the number of HLH cases due to ICI combined with anti-vascular therapy remains low, and further confirmation of the relevance is needed.
In our case, the patient had a history of psoriasis, and our case review found that 20.5% of patients with ICIrelated HLH had a history of autoimmune disease.Recent studies have demonstrated that pre-existing autoantibodies (including antithyroid [62], antinuclear [63] and other autoimmune-related antibodies [64]) are strong biomarkers for irAE, and autoimmune diseases such as rheumatoid arthritis and psoriasis have been shown to confer an elevated risk of irAEs [65].We suggest that, in HLH as in other irAEs, patients with autoantibodies and autoimmune diseases are likely to be at a higher risk of developing the disease.
Among the symptoms exhibited by the patient, fever remains the most common.Interestingly, in terms of laboratory tests, there is a difference from our case compared to most of the reported cases, as they often experienced a drop in platelets as the first symptom.However, our case showed granulocyte deficiency as the initial abnormal laboratory test, and the platelet count remained normal from the onset of HLH to the end.No laboratory test results similar to ours have been found in the cases collected so far.
Regarding the treatment of HLH, patients (n = 25 or 48%) used steroids alone or in combination with antibiotics.Typically, patients with HLH refractory to steroids are treated with immunosuppressive drugs such as cyclophosphamide, or with etoposide, a drug that may even be used as a front-line treatment in immunosuppressed patients [66].IL-6 inhibition with specific anti-IL-6 receptor antibodies, such as tocilizumab, has proven highly effective against cytokine release syndrome [67].One study suggested that IL-6 blockade administered alongside ICIs can ameliorate irAEs while enhancing the antitumoral effect of ICIs [68].In the 10 cases we collected where tocilizumab was used, only 1 death occurred, which may indicate the effectiveness of tocilizumab in treating HLH.However, confirmation through larger-scale case information is needed.
The prognosis of ICI-associated HLH is relatively favorable, with only 15.3% mortality in the pooled cases, which is much better than the 41% mortality reported in other types of HLH [2].However, among the four patients who developed immune-associated encephalitis, we found three deaths, suggesting that immune-associated encephalitis is a significant factor contributing to a poor prognosis.
ICI-associated HLH is undoubtedly a challenging complication that clinicians strive to avoid.However, we observed that patients who recover from it may experience improved tumor control.In recent years, numerous studies have demonstrated that irAEs may lead to a better prognosis for tumor treatment [58], but the link between high-grade irAEs and tumor prognosis remains poorly understood.A retrospective study based on a small sample showed that patients with grade 3 or higher irAEs had longer overall survival (OS) than those with grade 1 or 2 irAEs [69].Additionally, the results of a recent study, which combined data from three large clinical trials, revealed that grade 3-4 irAEs had a favorable prognostic effect over time compared to patients without irAEs [70].According to our summary data, ICI-associated HLH tended to have a very positive effect on tumor outcome, with varying degrees of tumor regression and stabilization in 80% of cases with available prognosis information.This effect was reported to persist after a period of follow-up in 8 cases, which aligns with our own case suggesting that recovery from ICI-associated HLH can have a significant impact on tumor tissue.This phenomenon was first proposed in 2016 by M Takeshita et al. [10], who discovered the coincidence of immunotherapy-associated hemophagocytic syndrome and rapid tumor regression.However, the exact mechanism still requires further investigation, as the number of cases is small.Nonetheless, the exact mechanism behind this observation warrants further investigation, given the limited number of cases currently available for study.It is crucial to continue researching this topic, taking into account that the primary goal of medical professionals is to avoid complications like ICI-associated HLH and provide the most effective and safe treatment for patients.
In conclusion, ICI-associated HLH may occur in any ICI and in any cancer, and combined anti-vascular therapy and the presence of a history of autoimmune disease may increase the incidence of HLH, with complete recovery in most cases of HLH after steroid-based therapy, and may have a favourable impact on tumour outcome.

Conclusions
HLH, a relatively understudied complication, tends to occur more frequently in patients with a history of autoimmune diseases.Our findings indicate that recovery from HLH during treatment may correlate with improved survival outcomes, underlining the need for further research in this area.
) meet Hscore > 169 or meet at least five of the eight criteria in HLH-2004.(Thespecific search formula and flowchart are shown in Supplemental data 2).

Fig. 2
Fig. 2 Bone marrow smear diagnosis and flow cytometry detection.A Hemophagocytosis in bone marrow and intracytoplasmic orthochromatic normoblast (black arrow); Wright-Giemsa staining, magnification of 400 ×.B Hemophagocytosis in bone marrow and intracytoplasmic granulocyte (black arrow); Wright-Giemsa staining, magnification of 400 ×.C Ratio of granulocytes, monocytes and lymphocyte in bone marrow detected by flowcytometry.D The percentage of T lymphocytes to total lymphocytes in bone marrow detected by flow cytometry.E The proportion of CD4 + T lymphocytes and CD8 + T lymphocytes to T lymphocytes

Fig. 3
Fig. 3 Leukocytes, absolute neutrophil count, hemoglobin and temperature trends during treatment.The X-axis represented the number of days from the patient received pembrolizumab and bevacizumab treatment; The left blue Y-axis responded to the value of leukocytes and absolute neutrophil count; The left green Y-axis responded to the patient's temperature value; The right red Y-axis responded to the patient's hemoglobin value.IG: immunoglobulin MP: methylprednisolone

Fig. 4
Fig. 4 ICI usage and associated clinical data.A Counts of various ICIs administered.B Number of cases across different tumor types.C Patient outcomes post-ICI treatment.D Tumor-specific responses following ICI therapy

Table 1
Context of HLH

Table 2
Clinical characteristics of HLH, treatment and outcome

Table 2 (continued) Clinical features Biological results Intervention outcome Other immune-related toxicities Rechallen-ge with ICI Tumor-Specific outcome Year country Ref
BM bone marrow, CR complete response, PD progressive disease, PR partial response, SD stable disease, Abs antibiotics, MTP methylprednisolone